Synthesis of a Lipopolysaccharide Designed to Conjugate Haptens and Proteins to Erythrocytes for Use as Target Cells in Passive Hemagglutination and Hemolytic Assays

Abstract

The synthesis of a reagent that is capable of conjugating a wide variety of haptens and proteins to cell membranes is presented. A very large chemically reactive glucose polymer (i.e., oxidized dextran), to which fatty acid side chains of myristic acid have been added, covalently binds to amino groups of haptens or proteins forming a Schiff base. The protein or hapten myristoyl dextran compounds attach directly to cell membranes when incubated with erythrocytes. The attachment of the ligands to the cell presumably occurs via the intercalation of the lipid portion of the myristoyl dextran compounds into the hydrophobic lipid bilayer of the plasma membrane. The conjugation of haptens and proteins to plasma membranes via this synthetic lipopolysaccharide reagent has several significant advantages over other coupling procedures: 1) conjugation of ligands occurs rapidly at conditions that are physiologic with respect to the cell, 2) both haptens and proteins can be conjugated via the same procedure, 3) the conjugating reagent, i.e., hapten-myristoyl dextran or protein-myristoyl dextran, is chemically stable and can be stored for extended periods of time at 4°C, 4) target cells prepared via myristoyl dextran detect picogram levels of antibody in hemolytic assays without any detectable nonspecific lysis, and 5) a very small amount of the hapten or protein myristoyl dextran reagent is required to produce target cells (i.e., as little as 1 to 10 µg of hapten). The following compounds have been successfully conjugated to cell membranes by using myristoyl dextrans as the linking reagent; N-ε-DNP-lysine, 3-aminopyridine, 4-aminopyridine, 4-aminobenzenearsonate, 4-aminobenzoate, 4-aminophthalate, 5-aminoisophthalate, Bence Jones protein, human γ-globulin, bovine γ-globulin, rabbit anti-human Fab antibody, and mouse anti-phthalate antibody. The effective use of myristoyl dextran-conjugated erythrocytes as target cells in the passive hemagglutination assay, the hemolytic spot test, the Jerne plaque assay, and an antigen-mediated hemolytic assay is established and the sensitivity of the target cells is quantitatively compared in these assays to target cells prepared by other commonly used procedures.